This is the second year of my research activities in NEI. I am continuing the effort to build a competitive genetic research labortory. Here I summaries the progress in research activities. 1. To analyze the PAX2 gene structure in patients collected in earlier studies: This is a continuation of my clinical ophthalmic research from Minnesota and collaboration. I developed a clinical DNA testing of PAX2 gene for Renal-coloboma Syndrome and have been collecting tested samples for genetic heterogeneity analysis. After I joined NEI, I have successfully recruited a contractor in April, 2009 to continue the project. We have developed an assay to analyze the coding region for genomic insertion/deletion using quantitative realtime PCR technology. We analyzed PAX2 gene 12 exons in 41 DNA samples. These patients had symptoms of Renal-coloboma syndrome. Coding region sequences were analyzed at University of Minnesota. We have not found any evidence of whole exonal insertion or deletion in these samples. We are extending this study to the PAX2 promoter region and other gene loci. 2. To analyze gene mutation in eye-related diseases: Developing new sequencing tests We are also developing sequence assays in several newly identified genes related to RP. We are sequencing EYS gene coding region in several RP patients. We are also sequencing PSTAN12 gene in several FEVR patients. We hope that we can also extend the assays into clinical testing protocols after validations. The lab has been working on several projects in collaboration with NEI intramural laboratories. Collaboration with Yuri Sergeev for Protein Modeling In our clinical testing, we continue to identify de novo mutations in the XLRS1 gene. We analyzed these mutations for functional consequences and its likely mechanism of inheritance with Yuri Sergeev in our branch. We presented a poster in the 2010 ARVO meeting. Collaboration with Dr. Sievings lab in a non-human primate model for Best Syndrome. Dr. Sievings lab identified a monkey with Best Syndrome. We systemically analyzed four normal monkeys as controls and the affected monkey for chromosomal abnormalities by Karyotyping and aCGH. Using candidate gene approach we have analyzed two genes for mutations that may cause macular dystrophy by DNA sequencing, RNA expression analysis and molecular cloning. We presented a poster in the 2010 Summer Poster Day. Collaboration with Central Serous choriodetinopathy (CSC) working group for genotyping in CSC patients We recently joined this collaboration leaded by Dr. Amy Chiu and starting a development of gene sequencing for a candidate gene, CLCN5. We have sequenced five CSC patients DNA samples in the coding region of CLCN5 gene. We did not find any sequence variation. Collaboration with Dr. Brian Brooks lab for CHM mutation analysis in Chorioderemia patients We are collaborating with Dr. Brooks lab to re-install a sequencing assay for CHM gene. We are optimizing the testing conditions and will start to work on patients soon. 3. To analyze promoter and intronic genomic abnormalities in Tyrosinase gene: Collaboration with Dr. Vincent Bearing (NCI) to analyze TYR gene expression This is also a continuation of my clinical research from Minnesota and collaboration with Dr. Bearing from NCI/NIH. I have been collecting tested samples for genetic heterogeneity analysis of TRY in Ocular-Cutaneous Albinism. After I joined NEI, I had a collaborator temporarily worked for me on this project in the summer 2009. He helped to establish several molecular procedures to analyze the promoter region of TYR gene and established collaboration with Dr. Bearings lab. I just had a research fellow started in January 2010 to continue this study. Right now, we are working on optimization of methylation analysis technologies. We hope that we will start a systemic analysis in patients with OCA1 very soon. Retinal resequencing chip analysis in 20 patients with isolated eye abnormalities: This project started several years ago in OGFVB. There were many personnel turnover in past years. The project was behind the schedule. However, there was a demanding from eyeGENE program that many of registered patients could not be assigned a genetic testing because of uncertainty of target gene(s). Screening by Resequencing CHIP on 93 genes provided an opportunity for these samples on hold. When I joined eyeGENE operation, I was requested to continue the project and evaluate the potential use of this resequencing CHIP for those patients on hold. I also considered that it was my obligation and commitment to NEI DNA lab and eyeGENE working group. In addition, the best way to appreciate the efforts of all authors of this project was to make it work. With Dr. Miller and Dr. Sohrabys help, I was able to secure the support for the operation. I eventually recruited Dr. Jin Song, to join the project in February, 2009. With the helps from Dr. Swaroop and Matthew Brooks from his core facility, we were eventually established the testing protocol. We tested 24 samples to validate the testing system. We also validated the variations identified by CHIP using Sanger sequencing. We are ready to evaluate the clinical research utility by testing more isolated RP patients. We are working on data analysis procedures and manuscript is preparation.